The Functional Roles Of Cyclin Dependent Kinase 5 Cdk5 Activators In Neuronal And Muscle Cell Differentiation

Cyclin Dependent Kinase 5 provides a comprehensive and up-to-date collection of reviews on the discovery, signaling mechanisms and functions of Cdk5, as well as the potential implication of Cdk5 in the treatment of neurodegenerative diseases. Since the identification of this unique member of the Cdk family, Cdk5 has emerged as one of the most important signal transduction mediators in the development, maintenance and fine-tuning of neuronal functions and networking. Further studies have revealed that Cdk5 is also associated with the regulation of neuronal survival during both developmental stages and in neurodegenerative diseases. These observations indicate that precise control of Cdk5 is essential for the regulation of neuronal survival. The pivotal role Cdk5 appears to play in both the regulation of neuronal survival and synaptic functions thus raises the interesting possibility that Cdk5 inhibitors may serve as therapeutic treatment for a number of neurodegenerative diseases.

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Abstract: Cyclin–dependent kinase 5 (CDK5) is a proline-directed serine/threonine kinase. It plays roles in regulation of important cell processes such as neural development, glucose-inducible secretion and wound healing, and aberrant activation of CDK5 has been implicated in diseases such as Alzheimer’s disease, type-2 diabetes, and cancer metastasis. Our laboratory studies molecular mechanisms involved in regulation of CDK5, specifically the role of phosphorylation in controlling CDK5 activity. This work focused on characterizing the effect of phosphomimetic and non-phosphorylatable mutations on the evolutionarily conserved serine residues present in the cyclin binding region (the “PS46S47ALRE” helix) of CDK5. The cyclin binding region on CDK5 mediates its interaction with the activator protein, p35, which is a key requirement for CDK5 activation. While the work on the S47 residue was carried out by another graduate student in the lab, my work focused on the S46 residue and the double S46/S47 mutants. The desired mutations (S→A and S→D) were generated using polymerase chain reaction-based site-directed mutagenesis. My co-immunoprecipitation assays revealed that a phosphomimetic (S→D) change at position S46 and the double mutant (S46D/S47D) disrupted the binding of CDK5 and p35, suggesting that phosphorylation of these residues will likely result in the same outcome and hence inactivate CDK5. The S→A single and double mutants retained binding to p35 and were active. I also conducted differential centrifugation experiments to determine the effect of these mutations on the subcellular localization of the CDK5-p35 complex. The disruption of binding to CDK5 appeared to affect the subcellular localization and/or stability of p25 (the truncated product of p35), however, larger number of biological replicates is needed in order to interpret the data. Finally, I began to investigate the effect of these mutations on cell migration using a scratch wound-healing assay. Together, the work presented in this study suggests that phosphorylation of S46 and S47 is a potential post-translational mechanism to control CDK5 activity by regulating binding to its activator. Future work should focus on detection of phosphorylated S46 and S47 in vivo and on the identification of kinases that mediate phosphorylation of these residues.

Cyclin Dependent Kinase 5 provides a comprehensive and up-to-date collection of reviews on the discovery, signaling mechanisms and functions of Cdk5, as well as the potential implication of Cdk5 in the treatment of neurodegenerative diseases. Since the identification of this unique member of the Cdk family, Cdk5 has emerged as one of the most important signal transduction mediators in the development, maintenance and fine-tuning of neuronal functions and networking. Further studies have revealed that Cdk5 is also associated with the regulation of neuronal survival during both developmental stages and in neurodegenerative diseases. These observations indicate that precise control of Cdk5 is essential for the regulation of neuronal survival. The pivotal role Cdk5 appears to play in both the regulation of neuronal survival and synaptic functions thus raises the interesting possibility that Cdk5 inhibitors may serve as therapeutic treatment for a number of neurodegenerative diseases.

A high-affinity inhibitor protein called CIP, which can be produced by small truncations of p35, was earlier identified by Amin, Albers, and Pant. P35 is one of the physiological activators of cdk5, a member of the cyclin-dependent kinase family. These proteins are known to be associated with the hyperphosphorylation of specific neuronal proteins. This typically occurs in the case of neurodegenerative diseases such as Alzheimer s. In this paper the authors study in silico the binding mechanism of cdk5-p25 and cdk5-CIP complexes more in detail. This provides a better understanding of the inhibitory activity of the protein CIP. The authors use a geometry-based technique to verify the following hypothesis: p25 s truncation provides increased flexibility to CIP, and hence CIP is able to conform better to cdk5 interface than p25 is. Therefore CIP is expected to bind to cdk5 more easily than p25 and prevent it from reaching its active conformation. The authors' in silico study is based on a geometry-based alignment algorithm. The algorithm is capable of efficiently aligning two protein conformations with respect to their interfaces, which are represented as point sets. The algorithm is based on biochemical criteria as well as geometrical ones.

"Loss-of-function mutations in the alkali cation/proton exchanger SLC9A6/NHE6 cause Christianson Syndrome (CS), a rare X-linked genetic disorder characterized by moderate to severe intellectual disability, epilepsy, ataxia, autistic behavior, and neurodegeneration. NHE6 is widely expressed but is especially abundant in nervous tissue where it localizes to vesicles along the recycling endosomal pathway of neurons and glia cells. Loss of NHE6 activity in cell and animal model systems results in excessive endosomal acidification, impaired cargo trafficking and signal transduction, as well as degeneration of certain neurons, though the molecular mechanisms underlying these phenomena are not fully resolved. We hypothesized that NHE6 mediates some of its effects through interactions of its C-terminal regulatory domain with cytoplasmic ancillary proteins, and that loss of these interactions is a contributing factor in the pathogenesis of CS. To this end, a yeast two-hybrid screen of a human brain cDNA library was performed by our group and lead to the identification of cyclin-dependent kinase 5 (CDK5) as a putative interacting partner of NHE6. CDK5 is a serine/threonine protein kinase expressed in most tissues, but primarily active in neurons. Loss of CDK5 expression results in a disease phenotype which closely resembles that observed for NHE6, including neuronal death, reduced neurite outgrowth, and increased susceptibility to seizures. In this study, we confirmed the formation of a NHE6 and CDK5 complex by biochemical (co-immunoprecipitation and glutathione S-transferase pull-down assays) and cell imaging (dual-labelling confocal microscopy and proximity ligation assay) analyses using Chinese hamster ovary AP-1 and neuroblastoma SH-SY5Y cells. CDK5 did not directly phosphorylate NHE6 in vitro despite the presence of a CDK5-like phosphor-acceptor site (S606PQA), though genetic manipulation of this site modestly affected CDK5 binding. Preliminary data from subcellular fractionation experiments suggest that NHE6 expression enhances CDK5 expression at the plasma membrane. However, the interaction between CDK5 and NHE6 is reduced in the presence of p35, an important activator of CDK5 that is tethered to the plasma membrane. Therefore, we propose that NHE6 serves as a scaffold for the endosomal delivery of CDK5 to the plasma membrane. There, CDK5 dissociates from NHE6 upon binding p35 and is now primed to activate neighboring effectors important for neuronal function"--

Parkinson's disease (PD) results primarily from the death of dopaminergic neurons in the substantia nigra. Current PD medications treat symptoms; none halt or retard dopaminergic neuron degeneration. The main obstacle to developing neuroprotective therapies is a limited understanding of the key molecular mechanisms that provoke neurodegeneration. The discovery of PD genes has led to the hypothesis that misfolding of proteins and dysfunction of the ubiquitin-proteasome pathway are pivotal to PD pathogenesis. Previously implicated culprits in PD neurodegeneration, mitochondrial dysfunction, and oxidative stress may also act in part by causing the accumulation of misfolded proteins, in addition to producing other deleterious events in dopaminergic neurons. Neurotoxin-based models have been important in elucidating the molecular cascade of cell death in dopaminergic neurons. PD models based on the manipulation of PD genes should prove valuable in elucidating important aspects of the disease, such as selective vulnerability of substantia nigra dopaminergic neurons to the degenerative process.